Project Summary Ubiquitin-mediated protein degradation is essential to maintaining proteostasis, thereby controlling diverse cellular processes. The specificity of protein ubiquitination is largely determined by recognition of short peptide motifs known as degrons in protein substrates by E3 ubiquitin ligases. F-box proteins serve as substrate- recognition components of the Skp1?Cul1?F-box-protein (SCF) E3 superfamily. The F-box protein FBXO31 mediates cyclin D1 degradation to induce G1 arrest after DNA damage. The C-terminal region of cyclin D1 contains sequences rich in Pro, Glu/Asp, Ser and Thr. This so-called PEST motif is enriched in highly dynamic disordered regions of eukaryotic proteins and serves as degradation signal for many unstable proteins. We have recently determined the X-ray crystal structure of a C-terminal PEST-containing cyclin D1 peptide bound to Skp1?FBXO31. The structure reveals that specific recognition is achieved by an intricate inter-molecular interaction network involving as well the cyclin D1 carboxyl tail that occupies an open surface cavity in the ?- barrel motif of FBXO31. We show that this PEST degron mediates the interaction of cyclin D1 with FBXO31 for ubiquitination in vitro and proteolysis in cells. We hypothesize that FBXO31 is responsible for recognition of C- terminal PEST-containing substrate degrons through a novel DesCEND (destruction via C-end degrons) pathway. By identifying and characterizing substrates of FBXO31 at both a systems level and the molecular level, we aim to elucidate the complementary structural determinants of the FBXO31?degron pair that confer substrate specificity and dictate protein recognition and fate. To achieve our goals, we have developed the following specific aims to: 1) identify and characterize C-end substrate degrons targeted by FBXO31 in human proteins; 2) characterize the structural and biochemical properties of candidate FBXO31 degrons; and 3) characterize endoproteolytic cleavage-generated FBXO31 degrons.